Molecular advances have provided greater understanding of skeletal muscle diseases, such as muscular dystrophy (MD), beginning with the discovery of the dystrophin gene and its gene product. There are nine types of MD, each a genetic degenerative disease primarily affecting voluntary muscles. Duchenne muscular dystrophy (DMD) affects 1 in 3500 live male births. The progressive muscle weakness and degeneration usually lead to loss of ambulation and wheelchair dependency in the early teens. Death occurs anytime after age 18 due to respiratory infection usually complicated by cardiac failure. The disease is caused by mutations in the dystrophin gene, which encodes a large (427 kDa) cytoskeletal protein in both skeletal and cardiac muscle. The dystrophin gene is the largest gene identified to date. It shows one of the highest sporadic mutation rates, with 1 of every 10,000 germ cells showing de novo mutations. Thus, the high incidence (1/3500), de novo mutations, early morbidity and fatality, and the lack of effective treatment require urgency in the search for novel therapeutics. Since DMD was described more than 140 years ago, the life-span of the DMD patient has only been marginally prolonged and the quality of life may not have changed significantly improved despite tremendous advances in medicine. Few treatments have been added to the current repertoire. In the muscular dystrophies, only corticosteroids have altered the natural history of disease and indeed the mechanism of action of corticosteroids in DMD remains unknown. In fact, the known myopathic effects of steroids might predict a deleterious effect but instead there is a paradoxical response resulting in an increase in muscle mass contrasting with the effects in normal muscle. Unfortunately the benefit of steroids comes at a high cost in terms of side effects (bone loss, cataracts, delayed puberty, weight gain and hypertension) providing compelling reasons to find other approaches.
Amyotrophic Lateral Sclerosis (ALS) is another disease that results in loss of muscle and/or muscle function. First characterized by Charcot in 1869, it is a prevalent, adult-onset neurodegenerative disease affecting nearly 5 out of 100,000 individuals. ALS occurs when specific nerve cells in the brain and spinal cord that control voluntary movement gradually degenerate. Within two to five years after clinical onset, the loss of these motor neurons leads to progressive atrophy of skeletal muscles, which results in loss of muscular function resulting in paralysis, speech deficits, and death due to respiratory failure. The genetic defects that cause or predispose ALS onset are unknown, although missense mutations in the SOD-1 gene occurs in approximately 10% of familial ALS cases, of which up to 20% have mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1), located on chromosome 21. SOD-1 normally functions in the regulation of oxidative stress by conversion of free radical superoxide anions to hydrogen peroxide and molecular oxygen. To date, over 90 mutations have been identified spanning all exons of the SOD-1 gene. Some of these mutations have been used to generate lines of transgenic mice expressing mutant human SOD-1 to model the progressive motor neuron disease and pathogenesis of ALS.
Another musculoskeletal disorder, inclusion body myositis (IBM), was originally named by Yunis and Samaha in 1971 to describe a patient with a chronic inflammatory myopathy who had intranuclear and intracytoplasmic tubular filaments within muscle fibers on electron microscopy. Many clinical and pathologic studies over more than three decades have supported this condition as a disorder distinct from other idiopathic inflammatory myopathies. Although incidence and prevalence statistics need further refinement, it is unequivocally the most common acquired muscle disease occurring after age of 50, with an estimated prevalence at 4-9:1,000,000. More men are affected than women by a ratio of greater than 2:1. Typically IBM is a sporadic disorder with insidious onset, and distinctive clinical and histopathological features (sIBM). Inflammation is prominent, helping to distinguish it from the group of inherited disorders (hIBM). These include autosomal recessive and dominant conditions with pathologic features resembling sIBM without inflammatory infiltration. Perhaps the best characterized hIBM is associated with mutations of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) [6].
The clinical features of sIBM include an average time of about 6 years from symptom onset to diagnosis. Difficulty with ambulation and frequent falls can be attributed to weak knee extensor muscles. Early involvement of the quadriceps and forearm flexor muscle compartment is typical. This is accompanied by a scooped-out appearance of the medial aspect of the forearms and thin, atrophic quadriceps muscles. Quadriceps atrophy and weakness is directly related to loss of safe ambulation. Frequent, sudden falls occur in three-fourths of cases and are the prime reason for wheelchair use. Only a minority are totally wheelchair dependent (˜15%). Mean time between symptom onset and wheelchair use ranged from 13±8 (6-32) years. Clinical dysphagia is common with estimates reaching as high as 66%. Finger flexor weakness can be detected early in the course of illness. About 40% of patients have facial muscle weakness. In the only prospective study of decline of muscle strength, the investigators documented a 4% decline in 6 months (0.66% per month) based on quantitative myometry using maximal isometric contraction testing. A similar rate of decline was also found in another study using manual muscle testing. Natural history studies document a fairly consistent, slow rate of decline in most patients with sIBM.
Therapy for sIBM has focused on the underlying inflammatory response with either immunosuppressive or immunomodulating drugs. Uncontrolled trials with corticosteroids, cyclophosphamide, chlorambucil, azathioprine and cyclosporin, and methotrexate fail to show convincing evidence of benefit. An initial pilot study of IVIG in IBM showed encouraging results but a subsequent randomized controlled trial with IVIG was negative. A prospective, double-blind, placebo-controlled, 6 month trial of weekly IM injections of 30 μg of β-interferon-1a showed no benefit. A recently published open-label trial using etanercept showed increased hand grip strength without any other functional benefits. Considering the complexity of pathogenic factors, it may be sometime before an effective treatment can target both autoimmune and myodegenerative factors. Attention has shifted to growth modulatory approaches. In a 3 month, randomized, placebo-controlled, crossover study of oxandrolone, a synthetic anabolic steroid, a borderline significant improvement in overall strength was reported, as measured by quantitative myometry. The study was limited by small sample size and short duration of treatment. Exercise is another means of growth modulation and this has been tried in inflammatory muscle diseases. An increase in isometric torque and an improvement in functional activities was reported in five patients with sIBM following a 12-week isotonic training program for the knee extensors and flexors and the elbow and wrist flexors. The small sample size disallows any conclusion but exercise had no deleterious effects based on muscle biopsies performed pre- and post-exercise.
Research has shown that skeletal muscle utilizes a regulatory mechanism to control tissue mass. In a screen for novel members of the transforming growth factor-β (TGF-β) superfamily of growth and differentiation factors, myostatin [previously called growth and differentiation factor-8 (GDF-8)] was identified and has subsequently been shown to be a negative regulator of muscle formation. Myostatin is expressed in the myotome compartment of developing somites at E9.5 with expression continuing throughout adulthood, predominantly in skeletal muscles and adipose tissue. Myostatin is synthesized in a precursor form that undergoes two proteolytic processing events to remove the N-terminal signal sequence and the C-terminal fragment, which possess receptor-binding activity. Following proteolytic processing, the propeptide and the disulfide linked C-terminal dimer remain bound noncovalently in a latent complex. Myostatin can be activated by dissociation of the propeptide after proteolytic cleavage by a metalloproteinase of the bone morphogenic (BMP)/tolloid family. The dissociated C-terminal fragment is thus the biologically active species. For a review of the biosynthesis and signaling pathway of myostatin, see Lee, Ann Rev Cell Dev Biol, 20: 61-86 (2004).
Myostatin is conserved among species, especially in its C-terminal fragment which is identical across human, rat, murine, porcine, turkey and chicken species. Mutations within myostatin have been shown to be linked to the double muscling phenotype in cattle [Grobet et al., Nat Genet, 17: 71-74 (1997); Kambadur et al., Genome Res, 7:910-915 (1997); and McPherron and Lee, Proc Natl Acad Sci USA, 94:12457-12461 (1997)] and gross muscle hypertrophy in human subjects [Schuelke et al., N Eng J Med, 350: 2682-2688 (2004)]. Forced muscle atrophy has even been achieved with recombinant myostatin administration or over-expression of myostatin [Zimmers et al., Science, 296(5572): 1486-1488 (2002)]. Histology of muscles from myostatin null mice shows increased muscle mass resulting from hyperplasia and hypertrophy of the muscle with less fat and connective tissues. The hypothesis that it may be beneficial to block, remove, or reduce myostatin to promote regeneration and reduce fibrosis in MD has been explored in animal studies. Wagner et al., Ann Neurol, 52: 832-836 (2002) describes data obtained from crossing myostatin null mutant mice with mdx mice (which are models for dystrophin deficiency) showing that mdx mice lacking myostatin were stronger and more muscular than their mdx counterparts. In addition, Bogdanovich et al., Nature, 420: 418-421 (2002) report that when a neutralizing antibody to myostatin was administered to 4 week old mdx mice by intraperitoneal injection, an increase in body weight, muscle mass, muscle size and absolute muscle strength along with a significant decrease in muscle degeneration and concentrations of serum creatine kinase was observed. Similarly, Whittemore et al., Biochem Biophys Res Commun, 300: 965-971 (2003) describes that myostatin neutralizing antibodies increase muscle mass in adult mice. Tobin and Celeste, Curr Opin Pharma, 5: 328-332 (2005) reviews the myostatin pathway as well as studies testing the effects of reducing myostatin expression/activity.
Another review article, Wagner, Curr Opin Rheumatol, 17: 720-724 (2005), lists various therapeutic approaches of inhibiting myostatin that have been considered for treating human disease. For example, Wyeth has developed a humanized, anti-myostatin antibody called MYO-029 for clinical trials for treatment of muscular dystrophy in adult patients. The review article states the antibody or similar agent will hopefully be tested in other indications such as inflammatory myopathies, cachexia and sarcopenia. The author also notes that a number of endogenous inhibitors of myostatin, including the myostatin propeptide, follistatin, FLRG and GASP-1 could be modified for use as therapeutic agents. The review refers to two articles describing the effects of modified propeptide on muscle in mice, Wolfman et al., Proc Natl Acad Sci US, 100: 15842-15846 (2003) and Bogdanovich et al., FASEB J, 19: 543-549 (2004).
The Wagner review article states that there is significant data that follistatin is an in vivo inhibitor of myostatin and refers to the results of studies described in Lee and McPherron, Proc Natl Acad Sci USA, 98(16): 9306-9311 (2001) and Amthor et al., Developmental Biol., 270: 19-30 (2004) to support that statement. Follistatin is a secreted protein that inhibits the activity of TGF-β family members such as GDF-11/BMP-11. Follistatin-344 is a follistatin precursor that undergoes peptide cleavage to form the circulating Follistatin-315 isoform which includes a C-terminal acidic region. It circulates with myostatin propeptide in a complex that includes two other proteins, follistatin related gene (FLRG) and GDF associated serum protein (GASP-1). Follistatin-317 is another follistatin precursor that undergoes peptide cleavage to form the membrane-bound Follistatin-288 isoform. The DNA and amino acid sequences of the follistatin-344 precursor are respectively set out in SEQ ID NOs: 3 and 4. The Follistatin-288 isoform, which lacks a C-terminal acidic region, exhibits strong affinity for heparin-sulfate-proteoglycans, is a potent suppressor of pituitary follicle stimulating hormone, is found in the follicular fluid of the ovary, and demonstrates high affinity for the granulose cells of the ovary. The testis also produce Follistatin-288. The DNA and amino acid sequences of the follistatin-317 precursor are respectively set out in SEQ ID NOs: 5 and 6. Lack of follistatin results in reduced muscle mass at birth.
In the experiments described in the Lee and McPherron article, follistatin was over-expressed in transgenic mice. The mice showed increased muscling resulting from a combination of hyperplasia (increased muscle fiber number) and hypertrophy (increased muscle fiber diameter). The article proposes that follistatin binds the C-terminal dimer of myostatin and, in turn, inhibits the ability of myostatin to bind to activin type II receptors. Transgenic mice expressing high levels of myostatin propepetide or a dominant-negative form of activin type II receptor (Act RIIB) were also shown to exhibit increased muscle mass in the article.
The Amthor et al. article is stated to report that follistatin directly binds myostatin with high affinity, is co-expressed with myostatin in somites and prevents myostatin-mediated inhibition of limb muscle development in chick embryos. Indicating that the inhibitory effects of follistatin are not specific to myostatin evening in regard to muscle growth, the Wagner review article alternatively indicates that FLRG and GASP-1, which bind to and inhibit circulating myostatin, may prove to be specific inhibitors of myostatin for therapeutic use. FLRG is a protein that exhibits homology to a 10-cysteine repeat in follistatin. Hill et al., J Biol Chem, 277(43): 40735-40741 reports that FLRG binds circulating myostatin in vivo.
Yet another review article addressing the regulation of muscle mass by myostatin and clinical implications is Lee, Annu Rev Cell Dev Biol., 20: 61-86 (2404).
Adeno-associated virus (AAV) is a replication-deficient parvovirus, the single-stranded DNA genome of which is about 4.7 kb in length including 145 nucleotide inverted terminal repeat (ITRs). The nucleotide sequence of the AAV serotype 2 (AAV2) genome is presented in Srivastava et al., J Virol, 45: 555-564 (1983) as corrected by Ruffing et al., J Gen Virol, 75: 3385-3392 (1994). Cis-acting sequences directing viral DNA replication (rep), encapsidation/packaging and host cell chromosome integration are contained within the ITRs. Three AAV promoters (named p5, p19, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The two rep promoters (p5 and p19), coupled with the differential splicing of the single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. Rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992).
AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic. Moreover, AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo. Moreover, AAV transduces slowly dividing and non-dividing cells, and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is infectious as cloned DNA in plasmids which makes construction of recombinant genomes feasible. Furthermore, because the signals directing AAV replication, genome encapsidation and integration are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA such as a gene cassette containing a promoter, a DNA of interest and a polyadenylation signal. The rep and cap proteins may be provided in trans. Another significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus (56° to 65° C. for several hours), making cold preservation of AAV less critical. AAV may even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.
Multiple studies have demonstrated long-term (>1.5 years) recombinant AAV-mediated protein expression in muscle. See, Clark et al., Hum Gene Ther, 8: 659-669 (1997); Kessler et al., Proc Nat. Acad Sc. USA, 93: 14082-14087 (1996); and Xiao et al., J Virol, 70: 8098-8108 (1996). See also, Chao et al., Mol Ther, 2:619-623 (2000) and Chao et al., Mol Ther, 4:217-222 (2001). Moreover, because muscle is highly vascularized, recombinant AAV transduction has resulted in the appearance of transgene products in the systemic circulation following intramuscular injection as described in Herzog et al., Proc Natl Acad Sci USA, 94: 5804-5809 (1997) and Murphy et al., Proc Natl Acad Sci USA, 94: 13921-13926 (1997). Moreover, Lewis et al., J Virol, 76: 8769-8775 (2002) demonstrated that skeletal myofibers possess the necessary cellular factors for correct antibody glycosylation, folding, and secretion, indicating that muscle is capable of stable expression of secreted protein therapeutics.